How Can We Advance Integrative Biology Research in Animal Science in 21st Century? Experience at University of Ljubljana from 2002 to 2022.

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health.

Distinctive probiotic features share common TLR2-dependent signalling in intestinal epithelial cells.

The underlying mechanisms of probiotics and postbiotics are not well understood, but it is known that both affect the adaptive and innate immune responses. In addition, there is a growing concept that some probiotic strains have common core mechanisms that provide certain health benefits. Here, we aimed to elucidate the signalization of the probiotic bacterial strains Lactobacillus paragasseri K7, Limosilactobacillus fermentum L930BB, Bifidobacterium animalis subsp. animalis IM386 and Lactiplantibacillus plantarum WCFS1. We showed in in vitro experiments that the tested probiotics exhibit common TLR2- and TLR10-dependent downstream signalling cascades involving inhibition of NF-κB signal transduction. Under inflammatory conditions, the probiotics activated phosphatidylinositol 3-kinase (PI3K)/Akt anti-apoptotic pathways and protein kinase C (PKC)-dependent pathways, which led to regulation of the actin cytoskeleton and tight junctions. These pathways contribute to the regeneration of the intestinal epithelium and modulation of the mucosal immune system, which, together with the inhibition of canonical TLR signalling, promote general immune tolerance. With this study we identified shared probiotic mechanisms and were the first to pinpoint the role of anti-inflammatory probiotic signalling through TLR10.

Nutrihealth Study: Seasonal Variation in Vitamin D Status Among the Slovenian Adult and Elderly Population.

Several studies conducted around the world showed substantial vitamin D insufficiency and deficiency among different population groups. Sources of vitamin D in the human body include ultraviolet B (UVB)-light-induced biosynthesis and dietary intake, but people's diets are often poor in vitamin D. Furthermore, in many regions, sun exposure and the intensity of UVB irradiation during wintertime are not sufficient for vitamin D biosynthesis. In Slovenia, epidemiological data about vitamin D status in the population were investigated through a national Nutrihealth study-an extension to the national dietary survey SI.Menu (2017/18). The study was conducted on a representative sample of 125 adult (18-64 years) and 155 elderly (65-74 years old) subjects, enrolled in the study in different seasons. Their vitamin D status was determined by measuring the serum 25-hydroxy-vitamin D (25(OH)D) concentration. Thresholds for vitamin D deficiency and insufficiency were 25(OH)D levels below 30 and 50 nmol/L, respectively. Altogether, 24.9% of the adults and 23.5% of the elderly were found to be vitamin D deficient, while an insufficient status was found in 58.2% and 62.9%, respectively. A particularly concerning situation was observed during extended wintertime (November-April); vitamin D deficiency was found in 40.8% and 34.6%, and insufficient serum 25(OH)D levels were observed in 81.6% and 78.8%, respectively. The results of the study showed high seasonal variation in serum 25(OH)D levels in both the adult and elderly population, with deficiency being especially pronounced during wintertime. The prevalence of this deficiency in Slovenia is among the highest in Europe and poses a possible public health risk that needs to be addressed with appropriate recommendations and/or policy interventions.

Health Professionals' Knowledge of Probiotics: An International Survey.

The objective of this study was to survey health professionals to investigate their knowledge of probiotics. An online survey was conducted to gather data on the knowledge of health professionals. The online survey was distributed via email and social media platforms using snowball sampling. A total of 1066 health professionals (859; 80.6% female) from 30 countries responded to the survey. Most of the respondents evaluated their knowledge of probiotics as medium (36.4%) or good (36.2%). Only 8.9% of the respondents rated it as excellent. No statistical difference in knowledge was found between male and female health professionals. Over 80% of pharmacists, allied health professionals, medical doctors and dentists, and other health professionals knew the correct definition of probiotics as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host", whereas three quarters of registered nurses and midwives and less than two thirds of psychologists identified the correct definition. Statistically, more female than male health professionals knew the correct definition of probiotics. The most frequently recognized species of bacteria containing probiotic strains were Lactobacillus acidophilus (92%), Bifidobacterium bifidum (82%), and Lactobacillus rhamnosus (62%). The opinions on when it is best to take probiotics were different (χ2 = 28.375; p < 0.001), with 90.2% of respondents identifying that probiotics have beneficial effects if taken during antibiotic therapy, 83.5% for diarrhea, 70.6% for constipation, 63.3% before traveling abroad, and 60.4% for treating allergies. Almost 79% of health professionals involved in this study have advised their patients to use probiotics and 57.5% of the respondents wanted to learn more about probiotics. All things considered, health professionals have a medium level of knowledge of probiotics, which could be improved by the implementation of targeted learning programs. As probiotics have many beneficial effects in a wide range of health areas, health professionals need to adopt the use of probiotics in clinical practice.

Efficacy of Using Probiotics with Antagonistic Activity against Pathogens of Wound Infections: An Integrative Review of Literature.

The skin and its microbiota serve as physical barriers to prevent invasion of pathogens. Skin damage can be a consequence of illness, surgery, and burns. The most effective wound management strategy is to prevent infections, promote healing, and prevent excess scarring. It is well established that probiotics can aid in skin healing by stimulating the production of immune cells, and they also exhibit antagonistic effects against pathogens via competitive exclusion of pathogens. Our aim was to conduct a review of recent literature on the efficacy of using probiotics against pathogens that cause wound infections. In this integrative review, we searched through the literature published in the international following databases: PubMed, ScienceDirect, Web of Science, and Scopus using the search terms "probiotic" AND "wound infection." During a comprehensive review and critique of the selected research, fourteen in vitro studies, 8 animal studies, and 19 clinical studies were found. Two of these in vitro studies also included animal studies, yielding a total of 39 articles for inclusion in the review. The most commonly used probiotics for all studies were well-known strains of the species Lactobacillus plantarum, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus rhamnosus. All in vitro studies showed successful inhibition of chosen skin or wound pathogens by the selected probiotics. Within the animal studies on mice, rats, and rabbits, probiotics showed strong opportunities for counteracting wound infections. Most clinical studies showed slight or statistically significant lower incidence of surgical site infections, foot ulcer infection, or burn infections for patients using probiotics. Several of these studies also indicated a statistically significant wound healing effect for the probiotic groups. This review indicates that exogenous and oral application of probiotics has shown reduction in wound infections, especially when used as an adjuvant to antibiotic therapy, and therefore the potential use of probiotics in this field remains worthy of further studies, perhaps focused more on typical skin inhabitants as next-generation probiotics with high potential.

Determination of Biogenic Amines in Cheese by Ion Chromatography with Tandem Mass Spectrometry Detection.

A new method for determination of underivatized biogenic amines in cheese based on ion exchange chromatography coupled with tandem mass spectrometric detection was proposed. The method was applied to the analysis of 10 biogenic amines (trimethylamine, putrescine, cadaverine, histamine, 2-phenylethylamine, spermine, spermidine, tryptamine, agmatine, and tyramine) in different types of cheese. The amines were extracted only with water without any additional derivatization step or sample cleanup. This is a great advantage in terms of simplicity of sample pretreatment procedure compared with other currently existing methods in the literature. Biogenic amines were separated using cation exchange column, under gradient elution conditions by mixing formic acid (1.00 M) and deionized water. Detection was achieved using tandem MS/MS, with the instrument set into multiple reaction monitoring mode to ensure high specificity. The detection and quantification limits were in the ranges of 12-46 µg/L and 40-153 µg/L, respectively. The exceptions were spermidine and spermine, with detection limits of 0.8 and 5.4 mg/L, respectively. The linearity for most of the biogenic amines was from 10 µg/L up to 10 mg/L. The best recoveries were observed for trimethylamine, tyramine, and cadaverine, and were 89, 94, and 102%, respectively. The results showed that this method can be used for routine determination of biogenic amines in different types of cheeses as well other food matrices. It must be stressed that the proposed method is capable of determining 10 biogenic amines, including tyramine, which is reported to cause food intoxication commonly associated with cheeses.

Microbes in Infant Gut Development: Placing Abundance Within Environmental, Clinical and Growth Parameters.

Sound and timely microbial gut colonization completes newborn's healthy metabolic programming and manifests in infant appropriate growth and weight development. Feces, collected at 3, 30, and 90 days after birth from 60 breastfed Slovenian newborns, was submitted to microbial DNA extraction and qPCR quantification of selected gut associated taxa. Multivariate regression analysis was applied to evaluate microbial dynamics with respect to infant demographic, environmental, clinical characteristics and first year growth data. Early microbial variability was marked by the proportion of Bacilli, but diminished and converged in later samples, as bifidobacteria started to prevail. The first month proportions of enterococci were associated with maternity hospital locality and supplementation of breastfeeding with formulae, while Enterococcus faecalis proportion reflected the mode of delivery. Group Bacteroides-Prevotella proportion was associated with infant weight and ponderal index at first month. Infant mixed feeding pattern and health issues within the first month revealed the most profound and extended microbial perturbations. Our findings raise concerns over the ability of the early feeding supplementation to emulate and support the gut microbiota in a way similar to the exclusively breastfed infants. Additionally, practicing supplementation beyond the first month also manifested in higher first year weight and weight gain Z-score.

Effects of synbiotic fermented milk containing Lactobacillus acidophilus La-5 and Bifidobacterium animalis ssp. lactis BB-12 on the fecal microbiota of adults with irritable bowel syndrome: A randomized double-blind, placebo-controlled trial.

We conducted a randomized double-blind, placebo-controlled multicentric study to investigate the influence of a synbiotic fermented milk on the fecal microbiota composition of 30 adults with irritable bowel syndrome (IBS). The synbiotic product contained Lactobacillus acidophilus La-5, Bifidobacterium animalis ssp. lactis BB-12, Streptococcus thermophilus, and dietary fiber (90% inulin, 10% oligofructose), and a heat-treated fermented milk without probiotic bacteria or dietary fiber served as placebo. Stool samples were collected after a run-in period, a 4-wk consumption period, and a 1-wk follow-up period, and were subjected to real-time PCR and 16S rDNA profiling by next-generation sequencing. After 4wk of synbiotic (11 subjects) or placebo (19 subjects) consumption, a greater increase in DNA specific for L. acidophilus La-5 and Bifidobacterium animalis ssp. lactis was detected in the feces of the synbiotic group compared with the placebo group by quantitative real-time PCR. After 1wk of follow-up, the content of L. acidophilus La-5 and B. animalis ssp. lactis decreased to levels close to initial levels. No significant changes with time or differences between the groups were observed for Lactobacillus, Enterobacteriaceae, Bifidobacterium, or all bacteria. The presence of viable BB-12- and La-5-like bacteria in the feces resulting from the intake of synbiotic product was confirmed by random amplification of polymorphic DNA (RAPD)-PCR. At the end of consumption period, the feces of all subjects assigned to the synbiotic group contained viable bacteria with a BB-12-like RAPD profile, and after 1wk of follow-up, BB-12-like bacteria remained in the feces of 87.5% of these subjects. The presence of La-5-like colonies was observed less frequently (37.5 and 25% of subjects, respectively). Next-generation sequencing of 16S rDNA amplicons revealed that only the percentage of sequences assigned to Strep. thermophilus was temporarily increased in both groups, whereas the global profile of the fecal microbiota of patients was not altered by consumption of the synbiotic or placebo. In conclusion, daily consumption of a synbiotic fermented milk had a short-term effect on the amount and proportion of La-5-like strains and B. animalis ssp. lactis in the fecal microbiome of IBS patients. Furthermore, both synbiotic and placebo products caused a temporary increase in fecal Strep. thermophilus.

Inulin-type fructan fermentation by bifidobacteria depends on the strain rather than the species and region in the human intestine.

Inulin-type fructans (ITF) are known to cause a health-promoting bifidogenic effect, although the ITF degradation capacity of bifidobacteria in different intestinal regions remains unclear. The present study aims at offering new insights into this link, making use of a collection of 190 bifidobacterial strains, encompassing strains from gut biopsies (terminal ileum and proximal colon; mucosa-associated strains) and the simulator of the human intestinal microbial ecosystem (SHIME®; proximal and distal colon vessels; lumen-associated strains). A multivariate data analysis of all fermentation data revealed four clusters corresponding with different types of ITF degradation fingerprints, which were not correlated with the region in the intestine, suggesting that the degradation of ITF is uniform along the human intestine. Strains from cluster 1 consumed fructose, while strains from cluster 2 consumed more oligofructose than fructose. Higher fructose and oligofructose consumption was characteristic for clusters 3 and 4 strains, which degraded inulin too. In general, the mucosa-associated strains from biopsy origin seemed to be more specialized in the consumption of fructose and oligofructose, while the lumen-associated strains from SHIME origin displayed a higher degradation degree of inulin. Further, intra-species variability in ITF degradation was found, indicating strain-specific variations. The coexistence of different bifidobacterial strains with different ITF degradation fingerprints within the same intestinal region suggests cooperation for the degradation of ITF, with opportunities for cross-feeding on strain and/or species level.

Vitamin D Status and Its Determinants in Healthy Slovenian Pregnant Women.

BACKGROUND/AIMS: Vitamin D deficiency is a common underdiagnosed condition. The aim of this was to analyze the status of vitamin D and its determinants in healthy Slovenian pregnant women. METHODS: A total of 132 volunteer pregnant women completed a questionnaire including baseline demographics, food frequency, physical activities; anthropometrical measurements, body mass index and levels of 25-(OH)D in serum were performed during the third trimester, and dietary intakes were assessed during the 27-28th week of gestation. RESULTS: Vitamin D deficiency was present in 14% while insufficiency was present in 41% of women. The risk for inadequacy was higher in women older than 30 years (p = 0.01), in those with less frequent outdoor physical activity (p = 0.01) and in pregnancies during the low sun exposure season (p = 0.04). Insufficiency was not significantly more frequent in less educated women, unemployed and in those living in urban area. The median value of vitamin D from habitual dietary intake was 1.5 µg/day (range 0.1-13.4) and did not influence 25-hydroxyvitamin D level (p = 0.91). CONCLUSIONS: The prevalence of vitamin D inadequacy was 55% and was dependent on age, season and outdoor physical activities. The results suggest a discrepancy between vitamin D intake through habitual diet and the reference needs.

Comparison of a Web-Based Dietary Assessment Tool with Software for the Evaluation of Dietary Records.

BACKGROUND: Dietary assessment in clinical practice is performed by means of computer support, either in the form of a web-based tool or software. The aim of the paper is to present the results of the comparison of a Slovenian web-based tool with German software for the evaluation of four-day weighted paper-and-pencil-based dietary records (paper-DRs) in pregnant women. METHODS: A volunteer group of pregnant women (n=63) completed paper-DRs. These records were entered by an experienced research dietitian into a web-based application (Open Platform for Clinical Nutrition, OPEN, http://opkp.si/en, Ljubljana, Slovenia) and software application (Prodi 5.7 Expert plus, Nutri-Science, Stuttgart, Germany, 2011). The results for calculated energy intake, as well as 45 macro- and micronutrient intakes, were statistically compared by using the non-parametric Spearman's rank correlation coefficient. The cut-off for Spearman's rho was set at >0.600. RESULTS: 12 nutritional parameters (energy, carbohydrates, fat, protein, water, potassium, calcium, phosphorus, dietary fiber, vitamin C, folic acid, and stearic acid) were in high correlation (>0.800), 18 in moderate (0.600-0.799), 11 in weak correlation (0.400-0.599), while 5 (arachidonic acid, niacin, alpha-linolenic acid, fluoride, total sugars) did not show any statistical correlation. CONCLUSION: Comparison of the results of the evaluation of dietary records using a web-based dietary assessment tool with those using software shows that there is a high correlation for energy and macronutrient content.

Behavior of Staphylococcus aureus in culture broth, in raw and thermized milk, and during processing and storage of traditional Greek Graviera cheese in the presence or absence of Lactococcus lactis subsp. cremoris M104, a wild, novel nisin A-producing raw milk isolate.

This study was conducted to evaluate the behavior of Staphylococcus aureus during processing, ripening, and storage of traditional Greek Graviera cheese in accordance with European Union Regulation 1441/2007 for coagulase-positive staphylococci in thermized milk cheeses. Lactococcus lactis subsp. cremoris M104, a wild, novel nisin A-producing (NisA+) strain, also was evaluated as an antistaphylococcal adjunct. A three-strain cocktail of enterotoxigenic (Ent+) S. aureus increased by approximately 2 log CFU/ml when co-inoculated (at approximately 3 log CFU/ml) in thermized Graviera cheese milk (TGCM; 63°C for 30 s) with commercial starter culture (CSC) and/or strain M104 at approximately 6 log CFU/ml and then incubated at 37°C for 3 h. However, after 6 h at 37°C, significant retarding effects on S. aureus growth were noted in the order TGCM + M104 > TGCM + CSC = TGCM + CSC + M104 > TGCM. Additional incubation of TGCM cultures at 18°C for 66 h resulted in a 1.2-log reduction (P < 0.05) of S. aureus populations in TGCM + M104. The Ent + S. aureus cocktail did not grow but survived during ripening and storage when inoculated (at approximately 3 log CFU/g) postcooking into Graviera mini cheeses prepared from TGCM + CSC or TGCM + CSC + M104, ripened at 18°C and 90% relative humidity for 20 days, and stored at 4°C in vacuum packages for 2 months. A rapid 10-fold decrease (P < 0.05) in S. aureus populations occurred within the first 24 h of cheese fermentation. Reductions of S. aureus were greater by approximately 0.4 log CFU/g in CSC + M104 than in CSC only cheeses, concomitantly with the presence of NisA + M104 colonies and nisin-encoding genes in the CSC plus M104 cheeses and their corresponding microbial consortia only. A high level of selective survival of a naturally nisin-resistant EntC z S. aureus strain from the cocktail was noted in CSC + M104 cheeses and in coculture with the NisA + M104 strain in M-17 broth. In conclusion, although S. aureus growth inhibition is assured during Graviera cheese ripening, early growth of the pathogen during milk curdling and curd cooking operations may occur. Nisin-resistant S. aureus strains that may contaminate Graviera cheese milks postthermally may be difficult to control even by the application of the NisA + L. lactis subsp. cremoris strain M104 as a bioprotective adjunct culture.

Comparison of paper- and web-based dietary records: a pilot study.

BACKGROUND/AIMS: Paper-based dietary records (Paper-DR) can be replaced by web-based dietary records (Web-DR) in both epidemiological studies and clinical practice to reduce the time and logistic burden. We aimed to compare Paper-DR and Web-DR. METHODS: We compared the matching of different food items (n = 1,103) from Paper-DR and Web-DR for energy and 48 nutrients among 16 pregnant volunteers, with DR for the same individuals matched for the same 4 days. Paper-DR were coded into the web-based version (referred to as Paper-Web-DR) independently by the same research dietitian. The Wilcoxon signed-rank test comparing mean rank differences, Spearman's ρ to measure associations and Bland-Altman limits of agreement to evaluate the level of agreement between the two dietary methods across the range of parameters were used. Volunteers also completed an evaluation questionnaire regarding the user acceptability of Paper-DR and Web-DR. RESULTS: A high correlation between Paper-DR and Web-DR was noted. There were statistically insignificant differences among 45 nutrients, except for free sugars (p < 0.001), α-linolenic acid (p = 0.041), folate (p = 0.036) and pantothenic acid (p = 0.023). Volunteers found the Paper-DR equally time-consuming as the Web-DR. The majority of the volunteers (75%) preferred the Web-DR. CONCLUSIONS: Paper-DR and Web-DR were comparable across a range of nutritional parameters, with a few exceptions. The Web-DR was more convenient for the majority and has substantial logistic and cost advantages.

Improved Draft Genome Sequence of Probiotic Strain Lactobacillus gasseri K7.

Lactobacillus gasseri K7 is an isolate from infant feces and has in vitro and in vivo established probiotic properties. Here, we report the improved version of the draft genome sequence, which comprises 8 scaffolds (13 contigs), a total length of 1.99 Mb, and 1,841 predicted protein-coding sequences.

Association of dietary type with fecal microbiota in vegetarians and omnivores in Slovenia.

PURPOSE: The purpose of this study was to discover differences in the human fecal microbiota composition driven by long-term omnivore versus vegan/lacto-vegetarian dietary pattern. In addition, the possible association of demographic characteristics and dietary habits such as consumption of particular foods with the fecal microbiota was examined. METHODS: This study was conducted on a Slovenian population comprising 31 vegetarian participants (11 lacto-vegetarians and 20 vegans) and 29 omnivore participants. Bacterial DNA was extracted from the frozen fecal samples by Maxwell 16 Tissue DNA Purification Kit (Promega). Relative quantification of selected bacterial groups was performed by real-time PCR. Differences in fecal microbiota composition were evaluated by PCR-DGGE fingerprinting of the V3 16S rRNA region. Participants' demographic characteristics, dietary habits and health status information were collected through a questionnaire. RESULTS: Vegetarian diet was associated with higher ratio (% of group-specific DNA in relation to all bacterial DNA) of Bacteroides-Prevotella, Bacteroides thetaiotaomicron, Clostridium clostridioforme and Faecalibacterium prausnitzii, but with lower ratio (%) of Clostridium cluster XIVa. Real-time PCR also showed a higher concentration and ratio of Enterobacteriaceae (16S rDNA copies/g and %) in female participants (p < 0.05 and p < 0.01) and decrease in Bifidobacterium with age (p < 0.01). DGGE analysis of the 16S rRNA V3 region showed that relative quantity of DGGE bands from certain bacterial groups was lower (Bifidobacterium, Streptococus, Collinsella and Lachnospiraceae) or higher (Subdoligranulum) among vegetarians, indicating the association of dietary type with bacterial community composition. Sequencing of selected DGGE bands revealed the presence of common representatives of fecal microbiota: Bacteroides, Eubacterium, Faecalibacterium, Ruminococcaceae, Bifidobacterium and Lachnospiraceae. Up to 4 % of variance in microbial community analyzed by DGGE could be explained by the vegetarian type of diet. CONCLUSIONS: Long-term vegetarian diet contributed to quantity and associated bacterial community shifts in fecal microbiota composition. Consumption of foods of animal origin (eggs, red meat, white meat, milk, yoghurt, other dairy products, fish and seafood) and vegetarian type of diet explained the largest share of variance in microbial community structure. Fecal microbiota composition was also associated with participants' age, gender and body mass.

Colostrum of healthy Slovenian mothers: microbiota composition and bacteriocin gene prevalence.

Microbial communities inhabiting the breast milk microenvironment are essential in supporting mammary gland health in lactating women and in providing gut-colonizing bacterial 'inoculum' for their infants' gastro-intestinal development. Bacterial DNA was extracted from colostrum samples of 45 healthy Slovenian mothers. Characteristics of the communities in the samples were assessed by polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and by quantitative real-time PCR (qPCR). PCR screening for the prevalence of bacteriocin genes was performed on DNA of culturable and total colostrum bacteria. DGGE profiling revealed the presence of Staphylococcus and Gemella in most of the samples and exposed 4 clusters based on the abundance of 3 bands: Staphylococcus epidermidis/Gemella, Streptococcus oralis/pneumonia and Streptococcus salivarius. Bacilli represented the largest proportion of the communities. High prevalence in samples at relatively low quantities was confirmed by qPCR for enterobacteria (100%), Clostridia (95.6%), Bacteroides-Prevotella group (62.2%) and bifidobacteria (53.3%). Bacterial quantities (genome equivalents ml-1) varied greatly among the samples; Staphylococcus epidermidis and staphylococci varied in the range of 4 logs, streptococci and all bacteria varied in the range of 2 logs, and other researched groups varied in the range of 1 log. The quantity of most bacterial groups was correlated with the amount of all bacteria. The majority of the genus Staphylococcus was represented by the species Staphylococcus epidermidis (on average 61%), and their abundances were linearly correlated. Determinants of salivaricin A, salivaricin B, streptin and cytolysin were found in single samples. This work provides knowledge on the colostrum microbial community composition of healthy lactating Slovenian mothers and reports bacteriocin gene prevalence.

Cryotrap/SPME/GC/MS method for profiling of monoterpenes in cheese and their clustering according to geographic origin.

A variant of purge/cryotrap/thaw/static headspace Solid Phase Microextraction (SPME) was developed as a means for preconcentrating Volatile Organic Compounds (VOC) in cheese. An originally designed cryotrap partially filled with glass beads was employed that facilitated efficient flow-through of purging gas and trapping of the volatiles. In stopped-flow mode, thawing was allowed, and the same vessel was used for the exposure of the appropriate SPME fiber, effectively achieving double preconcentration. Gas chromatography/mass spectrometry (GC/MS) was subsequently employed to identify components and assess their relative chromatographic peak areas. Monoterpenes were chosen as a model group of substances, and their relative concentration profiles were evaluated as potential markers for the respective geographic origin. The procedure was tested on samples of five traditional Slovenian cheeses featuring Protected Designation of Origin (PDO): Tolminc, Mohant, Nanoski cheese, together with Bovski cheese and Karst Ewe's cheese. The dataset of the peak areas of nine prominent monoterpenes (α-pinene, camphene, α-phellandrene, ß-pinene, 3-carene, 2-carene, limonene, tricyclene, and γ-terpinene) in cheese samples showed clustering that relates the cheeses to the area of production. According to the silhouette metrics, four clusters were identified by partitioning around medoids (PAM) method. The latter packed data for Tolminc and Bovski cheese into a single cluster, closely reflecting the vicinity of their geographic origin, but classified correctly the rest of the data into separate clusters for all other cheeses.

Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes.

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.

Challenges in determining body fat in pregnant women.

BACKGROUND/AIMS: Determining body composition in pregnant women is challenging as not all of the existing applicable methodologies can be used during pregnancy and not all of the methods have been properly standardized. The aim of this study was to compare the existing anthropometric methods for the evaluation of body composition, especially in pregnant women. METHODS: One hundred forty-seven pregnant volunteers aged [average (SD)] 31 years (± 4) in gestational week 32 (± 3) provided information on their age and prepregnancy body mass. Their height, current mass, skinfold thicknesses, and limb circumferences were measured. The body density and fat percentage were calculated according to 17 different anthropometric equations obtained from the literature. Data were analyzed with ANOVA. RESULTS: For the same sample of pregnant women, the body fat percentages obtained using the existing anthropometric methods varied greatly (p < 0.0001) and ranged from 16% (± 5) to 38% (± 4); methods developed specifically for pregnant women yielded disturbingly large differences, with body fat values ranging from 16% (± 5) to 36% (± 6). CONCLUSIONS: This study revealed large discrepancies among anthropometric methods for body composition assessment in pregnant women. As the results from the same sample obtained with different existing equations are wide ranging, the existing methodologies should be examined and improved before they can serve as sources of information regarding the health status of pregnant women.